The goal of this project is the development of new antineoplastic agents directed against tubulin, a protein critical for cell division and for the maintenance of cellular morphology. Detailed mechanistic and structure-activity studies were performed with many classes of compounds. Comparative studies were begun with new structurally unique agents that resemble paclitaxel in their effects on microtubules in cellular and biochemical systems. These are the sponge-derived discodermolide and the fermentation products epothilone A and B. Studies were also initiated with the cyanobacterial product cryptophycin A, which binds in a unique site on the tubulin molecule. Ongoing structure-activity studies continued with natural and synthetic analogs of the antimitotic agents dolastatin 10, dolastatin 15, spongistatin 1, curacin A, and taxol. Characterization of interactions of the mammalian metabolite 2-methoxyestradiol with tubulin and tubulin polymer continued. A-ring modified reactive colchicine derivatives were shown to form covalent bonds to cys-354 as well as cys-239 of beta-tubulin. The ability of GDP to inhibit paclitaxel induced tubulin assembly was studied in detail, and we conclude that GTP in trace amounts must be required for paclitaxel-induced microtubule formation.